Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality and can occur in smokers as well as non-smokers. Components of the Hippo tumor suppressive pathway are altered in NSCLC and recent studies have shown that this pathway can be modulated by upstream signaling events from K-Ras and EGFR. We had found that YAP1 could induce Sox2 and EMT markers, promoting the growth and metastasis of NSCLC. We find that the non-canonical IkB kinase TBK1 (Tank Binding Kinase 1) could physically interact with YAP1 and phosphorylate it; further, depletion of TBK1 led to a marked elevation of YAP1 levels under normoxic conditions, but only in K-Ras mutant, but not EGFR-mutant, lung adenocarcinoma cells. This was specific to TBK1, since depletion of the closely related IKKe kinase did not elevate YAP1 levels. We find that the induction of YAP1 occurs at the level of protein stability, brought about by the methylation of arginine residues of YAP1 by CARM1 (PRMT4). CARM1 is overexpressed in a variety of cancers and high levels of CARM1 correlates with poor survival in NSCLC patients. Based on these results, we hypothesize that the regulation of YAP1 by TBK1 and CARM1 is a novel mechanism which significantly promotes the growth and metastasis of non-small cell lung cancer. Studies proposed in this application will characterize this regulation mechanistically using a variety of in vitro and in vivo analysis, including co-culture studies and syngeneic transplantation models combined with global analysis of gene regulation by arginine-methylated YAP1 protein. The physical interaction of YAP1 with TBK1, PRMT5 and CARM1 will be assessed in three different human lung cancer TMAs from low grade and high grade tumors that harbor various K-Ras mutations; such an analysis will shed light on whether the levels and physical interaction of YAP1 with these regulatory molecules affect the growth and progression of these tumors. ChiP-re-ChIP, ChIP-Seq and RNA-Seq analysis will be conducted on primary tumor samples and adjacent normal tissue to identify the downstream targets of arginine-methylated YAP1. YAP1 has been demonstrated to have significant immunosuppressive effects; downregulating YAP1 through the inhibition of CARM1 can be expected to enhance the efficacy of immune checkpoint inhibitors. In depth studies will be conducted on syngeneic mouse models to assess how CARM1 inhibitors alone or in combination, affects the anti-tumor activity of T cells. Further, the fact that CARM1 inhibitors are in clinical trials for hematological malignancies raise the possibility that their utility can eventually be extended to K-Ras mutant lung cancers. We propose to test the anti-cancer efficacy of the CARM1 inhibitor EZM2302 alone or in combination with the TBK1 inhibitor Amlexanox, the K-Ras G12C inhibitor AGM510 or the MEK inhibitor Trametinib or the PLK1/K-Ras. Given the established oncogenic role of CARM1 and YAP1 in various cancers including those of the pancreas and the lung, an elucidation of these novel regulatory modes of YAP1 function would lead to the identification of vulnerabilities that could potentially be targeted to combat K-Ras mutant lung adenocarcinomas.